Generation of lineage-restricted progenitor cells from differentiated cells

ABSTRACT

Method for reprogramming differentiated cells into lineage restricted progenitor cells is provided. The method may include contacting differentiated cells with inhibitors of tyrosine phosphatases and apoptosis to de-differentiate differentiated cells into lineage restricted progenitor cells.

GOVERNMENT RIGHTS

This invention was made with government support under federal grant no.AG027252 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The subject invention is directed to methods for generating lineagerestricted progenitor cells. Aspects of the method include generatingnew cells in a subject in need thereof, using the lineage restrictedprogenitor cells. In addition, reagents, devices and kits thereof thatfind use in practicing the subject methods are provided.

INTRODUCTION

Maintenance and repair of tissue is essential for survival ofmulticellular organisms. However, there is a decline in maintenance andrepair of tissue with aging and in certain diseases. In addition,mammals have a limited ability to regenerate damaged tissue or replacedead cells in a tissue as majority of cells in the tissue are in adifferentiated state with limited proliferative capacity.

Skeletal muscle represents a classic example of terminal differentiationwherein myogenic proliferating cells expressing Pax7 and MyoDpermanently withdraw from the cell cycle upon serum deprivation andphysiologically fuse into multinucleated myotubes expressing muscledifferentiation markers myogenin and eMyHC (Okazaki and Holtzer, ProcNatl Acad Sci USA 56, 1484-1490, 1966; Olson, Dev Biol 154, 261-272,1992; Rudnicki and Jaenisch, Bioessays 17, 203-209, 1995). Theregenerative capacity of muscle stem cells declines upon aging and incertain pathologies exemplified by Duchenne muscular dystrophy. Hencestudying reprogramming of terminally differentiated muscle cells totheir proliferating progenitors holds not only theoretical value but isalso therapeutically relevant. The reprogramming from myotubes tomyogenic precursor cells is particularly challenging since myogenicproliferating cells not only undergo post-mitotic arrest, but alsophysically fuse with each other to form multinucleated myotubes duringtheir terminal differentiation. Once these cells terminallydifferentiate, they are incapable of re-entering into mitosis even whenswitched to serum rich medium (Endo and Nadal-Ginard, Mol Cell Biol 6,1412-1421, 1986; J Cell Sci 111 (Pt 8), 1081-1093, 1998). In contrast,reserve cells (myoblasts which remain mono-nucleated upon serumwithdrawal) can re-enter cell cycle when switched back to themitogen-high serum rich growth medium (Carnac et al., Curr Biol 10,543-546, 2000; Friday and Pavlath, J Cell Sci 114, 303-310, 2001;Yoshida et al., J Cell Sci 111 (Pt 6), 769-779, 1998). Over-expressionof cyclin D1 and CDK4/6 or knocking down cell cycle inhibitors alone orin combination is insufficient for myotubes to enter mitosis (Latella etal., Mol Cell Biol 21, 5631-5643, 2001; Tiainen et al., Cell GrowthDiffer 7, 1039-1050, 1996). Studies in C2C12 cells have shown that afraction of myotubes derived from this cell line can de-differentiate inthe presence of newt extract, myoseverin, or when msxl or twist areover-expressed (Duckmanton et al., Chem Biol 12, 1117-1126, 2005;Hjiantoniou et al., Differentiation 76, 182-192, 2008; McGann et al.,Proc Natl Acad Sci USA 98, 13699-13704, 2001; Odelberg et al., Cell 103,1099-1109, 2000; Rosania et al., Nat Biotechnol 18, 304-308, 2000).However, the rare de-differentiated cells were not tested for theirability to contribute to muscle regeneration in vivo. Earlier work hasalso reported that C2C12 myotubes responsive to thrombin activated serumresponse factor triggers expression of immediate early genes but is notsufficient for S phase entry (Loof et al., Cell Cycle 6, 1096-1101,2007). Interestingly, the same group also demonstrated that H3K9di-methylation remains unperturbed in C2C12 myotubes in the presence ofserum as opposed to salamander myotubes which readily enter cellproliferation. A recent study has shown deletion in Ink4a locus in C2C12immortalized cell lines which provides an advantage to C2C12 cells toenter cell cycle upon knockdown of Rb. Knockdown of pRb in conjunctionwith Arf can induce cell cycle entry in primary myocytes but not inprimary myotubes where nuclei get arrested at the onset of mitosis(Pajcini et al., Cell Stem Cell 7, 198-213, 2010). Nevertheless, theprocess of de-differentiation of primary multi-nucleated myotubes isstill not well understood and most of the previous studies relied on theover-expression of exogenous genes. Some of the previous studies haveemployed single myocyte and myotube isolation and that can lead topreferential selection of those myotubes that survive such process anddoes not clear ambiguity of reserve cells which can come along withmyotubes. Sparse plating of myoblasts was also tried, but that preventsformation of multinucleated myotubes and limits the study to myocytes.

As such, there is a need for methods and reagents for generating lineagerestricted progenitor cells that may be used to provide differentiatedcells.

SUMMARY

Method for reprogramming differentiated cells into lineage restrictedprogenitor cells is provided. The method may include contactingdifferentiated cells with inhibitors of tyrosine phosphatases andapoptosis to de-differentiate differentiated cells into lineagerestricted progenitor cells.

The subject methods find use in generating muscle tissue in vitro andgenerating new muscle tissue in a subject in need thereof. In addition,reagents and kits thereof that find use in practicing the subjectmethods are provided.

In certain embodiments, the method for generating lineage-restrictedprogenitor cells from a differentiated cell, may include contacting adifferentiated cell with an effective amount of an agent that inhibitstyrosine phosphatases and an effective amount of an agent that inhibitsapoptosis under conditions sufficient for generation oflineage-restricted progenitor cells from the differentiated cell,wherein the differentiated cell and the lineage-restricted progenitorcells have the same lineage.

The differentiated cell used in the methods disclosed herein may be anydifferentiated cell, such as, those described in the present disclosure.In certain embodiments, the differentiated cell may be a myocyte and thelineage-restricted progenitor cells may be myogenic progenitor cells. Incertain aspects of the invention, the myocyte may be a myocyte selectedfrom the group consisting of a cardiomyoctyte, a smooth muscle myocyte,and a skeletal myocyte.

The agent that inhibits tyrosine phosphatases may be a small molecule.The agent that inhibits apoptosis may be a small molecule.

In some embodiments, the differentiated cell may be from a subject witha disease condition.

In certain cases, the contacting may be carried out ex vivo or in vivo.

The method may further include transferring the lineage-restrictedprogenitor cells to conditions that promote differentiation intodifferentiated cells of the same lineage as that of the differentiatedcell contacted in the contacting step.

In certain cases, the transferring may include transferring thelineage-restricted progenitor cells into a subject. The subject may bein need of tissue regeneration.

In certain instances, the subject may be suffering from loss of musclefunction and/or loss of muscle mass, and wherein the differentiated cellis a myocyte and the lineage-restricted progenitor cells are myogenicprogenitor cells.

Also provided is a method of screening for an agent that inhibitstyrosine phosphatases and an agent that inhibits apoptosis and mediatesgeneration of lineage-restricted progenitor cells from a differentiatedcell. The method may include contacting a differentiated cell with acandidate agent that inhibits tyrosine phosphatases and a candidateagent that inhibits apoptosis under conditions sufficient for generationof lineage-restricted progenitor cells from the differentiated cell,wherein the differentiated cell and the have the same lineage; anddetermining whether lineage-restricted progenitor cells are produced,wherein the presence of lineage-restricted progenitor cells indicatesthat the candidate agent that inhibits tyrosine phosphatases and thecandidate agent that inhibits apoptosis mediate generation oflineage-restricted progenitor cells from a differentiated cell. Thedifferentiated cell may be a myocyte and the lineage-restrictedprogenitor cells may be myogenic progenitor cells.

A kit for use in generation of lineage-restricted progenitor cells froma differentiated cell is also disclosed. The kit may include an agentthat inhibits tyrosine phosphatases and an agent that inhibitsapoptosis. In certain cases, the agent that inhibits tyrosinephosphatases and the agent that inhibits apoptosis are small molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures:

FIG. 1. Lineage marking of primary myotubes by Cre-Lox method. FIG. 1A.Schematic of the system. Wild type myoblasts (MB) derived from C57BL/6mice were infected with Ad-Cre and subsequently co-cultured with Lox-YFPMB obtained from Rosa 26-YFP reporter mice in differentiation medium(DM) to form myotubes. The fusion of these two populations of MB led tothe excision of stuffer sequence (excised floxed sequence) by Crerecombinase activity to give rise to lineage marked YFP expressingmyotubes (shaded). Self fusion among the two populations of MB will giverise to YFP negative myotubes (colorless). These lineage marked myotubeswere then used in de-differentiation studies. FIG. 1B. Fusion-dependent,Cre-Lox mediated labeling of myotubes upon co-culture of Ad-Cre MB withLox-YFP MB in DM. As described in FIG. 1A, wild type MB were co-culturedwith Lox-YFP MB (1:2 ratio) in DM to induce formation of myotubes.Endogenous YFP fluorescence in myotubes was observed by 72-96 hours asshown by epifluorescent images. In control infection with control Ad-RFPvirus, no YFP fluorescence was observed. No YFP expression was observedupon co-culture of Ad-Cre MB and Lox YFP MB in GM where myoblasts didnot undergo physiological fusion to form myotubes. FIG. 1C. Westernblotting to determine YFP expression using lysates from Lox-YFP MB,Ad-Cre MB co-cultured with Lox-YFP MB in GM and parallel in DM. YFPprotein was observed in the myotubes which arose from fusion of Ad-Creand Lox-YFP MB in DM. FIG. 1D. qRT-PCR analysis for YFP gene expression.RNA was extracted from Ad-Cre and Lox-YFP MB co-cultured in GM and in DMfor 96 hours to detect the levels of YFP and Cre recombinase by qRT-PCR.Data was normalized to internal control GAPDH. Error bars indicate meanand standard deviation, n=3. YFP mRNA levels was only observed inAd-Cre-Lox-YFP⁺ myotubes while Cre recombinase expressed in both theco-cultures of Ad-Cre and Lox-YFP MB in GM and in DM. Thefusion-dependent marking of myotubes was clearly and robustly mediatedby this adaptation of the Cre-Lox method, and no mononucleated cellsexpressed YFP.

FIG. 2. Immunodetection of YFP and muscle specific markers. FIG. 2A-D.YFP⁺ myotubes obtained after Cre-Lox fusion express muscledifferentiation marker and do not incorporate BrdU. Cre-Lox YFP⁺myotubes cultures were co-immunostained with muscle differentiationmarkers eMyHC (FIG. 2A), myogenin (FIG. 2B), p21 (FIG. 2C), and DNAsynthesis label BrdU (FIG. 2D) along with anti-YFP antibody.Representative images are shown.

FIG. 3. BpV with Q-VD de-differentiates the irreversibly-labeled YFP⁺myotubes to YFP⁺ proliferating mononucleated cells. FIG. 3A. Myotubede-differentiation strategy. MB infected with Ad-Cre were co-culturedwith Lox-YFP MB in DM for 4 days to give rise to YFP⁺ myotubes. Thesewere treated with 10 μM BpV+10 uM Q-VD in parallel with otherexperimental conditions for two days in DM. The treated myotubes werethen switched to myoblast GM which was replaced fresh every day. YFP⁺mononucleated cells were observed around day 10. De-differentiation ofYFP⁺ myotubes to YFP⁺ proliferative cells. FIG. 3B. YFP⁺ myotubescultures were treated with the BpV+Q-VD and photographed every day. Theaddition of BpV+Q-VD led to morphological changes and when switched toGM these cells expanded as YFP⁺ mononucleated cells in 72 hours (whitearrow shows YFP⁺ mononucleated cells). Representative high magnificationimages of de-differentiation experiment over the course of 10 days withlive Hoechst is shown by epifluorescent microscopy. FIG. 3C.No-treatment (Untreated: UT): Untreated YFP⁺ myotubes were grown insimilar conditions and did not show any de-differentiation events. Thesedata demonstrate that inhibitor mix is necessary and sufficient forde-differentiation of genetically labeled myotubes into expandingmononucleated cells. FIG. 3D. Reprogrammed YFP⁺ mononucleated cellsrapidly divide. Cre-Lox-YFP⁺ myotubes reprogrammed as depicted in FIGS.3B and 3C, were pulsed with BrdU for 24 hours and co-stained with antiYFP and BrdU antibodies. Arrows indicate representative BrdU⁺YFP⁺ cellsin treated conditions. Untreated cultures of YFP⁺ myotubes do not showany YFP⁺ mononucleated cells though BrdU incorporation is seen in nonYFP cycling mononucleated cells. Inset shows magnified images. FIG. 3E.Quantification of percent of BrdU⁺/YFP⁺ mononucleated cells out of totalnumber of YFP⁺ myotubes (shown are the mean and standard deviations, n=3p<0.05). Note that many reserve myoblasts re-entered cell cycle andincorporated BrdU in GM (both in the presence of BpV+Q-VD and in controluntreated cultures); these cells, however, were reliably distinguishedin our experiments by the absence of YFP.

FIG. 4. Genetically-labeled progeny of de-differentiated myotubes havefunctional and genetic attributes of muscle progenitor cells. FIG. 4A.Co-immunostaining of FACS-sorted, proliferating YFP⁺ mononucleated cellsfor (i) Pax7 and (ii) MyoD along with anti-YFP antibody was performedand representative images are shown. FIG. 4B. Histogram quantifies Pax7and MyoD expressing YFP⁺ mononucleated cells which represents mean andstandard deviation of three independent experiments. FIG. 4C.De-differentiated, FACS sorted, YFP⁺ cells were expanded in GM andcultured in DM for 96 hours where myoblasts typically form myotubes;cultures were co-immunostained with antibodies specific to YFP and tomyotube specific marker (i) eMyHC ii) myogenin, as well as (ii) the CDKinhibitor p21. FIG. 4D. Western blotting with antibodies specific forPax7, MyoD, eMyHC, p21, myogenin and YFP was performed using proteinextracts from Cre-Lox-YFP myotubes, de-differentiated YFP⁺ mononucleatedcells and re-differentiated YFP⁺ cells as indicated. Actin served asloading control. FIG. 4E. Gene expression analysis of muscledifferentiation markers. qRT-PCR data in log scale for Pax7, MyoD,myogenin, p21 and eMyHC depicts the relative gene expression ofre-differentiated myotubes to de-differentiated YFP⁺ cells. These datarepresent the mean and standard error for three independent experiments.

FIG. 5. Reprogrammed YFP⁺ proliferative cells contribute to in vivomuscle regeneration. FACS sorted YFP⁺ proliferating mononucleated cellswere expanded in GM and injected in cardiotoxin injured TibialisAnterior (TA) immuno-compromised NOD-SCID mice. 2-3 weeks later, TAmuscles were dissected out, sectioned at 10 μm and co stained with YFPand laminin to visualize YFP⁺ myofibers. Control buffer and Lox YFPmyoblast injected TA muscle did not show any YFP⁺ myofibers.

FIG. 6. Molecular analysis of reprogramming in genetically labeledmyotubes. Inhibitor mix treatment down regulates muscle differentiationmarker in Cre Lox-YFP myotubes. FIGS. 6A and 6B. 4 day oldAd-Cre-Lox-YFP myotubes were untreated/treated with inhibitor mix for 48hours, followed by immuno-detection of myogenin (a), p21 (b) and YFP(green), using antibodies specific for these proteins. Myogenin and p21were down-regulated in a subset of YFP⁺ myotubes (shown by whitearrows). Control myotubes did not change expression of muscledifferentiation markers. FIGS. 6C and 6D. The histogram quantifies thepercent of YFP⁺/myogenin⁺, YFP⁺/myogenin⁻ cells and YFP⁺/p21⁺ andYFP⁺/p21⁻ in the experiment shown in FIGS. 6A and 6B (n=3±S.D.;p***<0.001, p**<0.05). FIGS. 6E and 6F. Ad-Cre-Lox-YFP myotubesuntreated/treated with BpV+Q-VD for 48 hours were analyzed for proteinand mRNA levels. Protein lysates were subjected to western blotting forantibodies against p21, myogenin and eMyHC. Actin served as a loadingcontrol. q-RT-PCR was performed on RNA lysates for gene expression ofp21, p15, p16, myogenin and eMyHC. Data were normalized to GAPDH andrepresents mean and standard deviation of three independent experimentseach done in triplicates (n=3±S.D.; p***<0.001, p**<0.05). Untreatedsample was taken as 1.

FIG. 7. Inhibitor mix treatment modulates chromatin remodeling factorsand enzymes. FIGS. 7A and 7B. Clustergram analysis of chromatinremodeling factors and enzymes for Ad-Cre-Lox YFP myotubes treated anduntreated with BpV+Q-VD (inhibitor mix) for 48 hours using SABiosciences/Qiagen PCR arrays. 0.5 ug RNA isolated from threeindependent set of experiments of Ad-Cre-Lox YFP myotubes were reversetranscribed and gene expression profile monitored. FIGS. 7C and 7D.Histogram representation for few set of genes normalized by Hprt genelevels. Control untreated was taken as 1. (n=3±S.D.; p***<0.001,p**<0.05).

FIG. 8 shows Supplementary Table S1 that provides a complete list ofchromatin factor and enzyme genes modulated by inhibitor mix treatment.

FIG. 9 shows Supplementary Table S2 that provides a complete list ofchromatin factor and enzyme genes modulated by inhibitor mix treatment.

FIG. 10A-D. Representative images of time lapse microscopy are shownfrom labeling strategy captured by time lapse microscopy encompassingtotal number of 4 days from the co-culture of Cre and Lox YFP myoblaststo their fusion into multinucleated myotubes.

FIG. 11. Results demonstrate that the inhibition of apoptosis is afactor for the de-differentiation of multinucleated primary myotubes.FIGS. 11A, 11C and 11D: In the presence of BpV alone, myotubes did showapoptosis and few of them gave rise to YFP+ mononucleated cells albeitat very low frequency (˜1.18%) in comparison to inhibitor mix treatmentwhich augmented the de-differentiation frequency to around ˜12-13%. FIG.11B: In control experiments, no YFP⁺ mononucleated cells were observedin untreated YFP⁺ myotubes in the presence of Q-VD alone.

FIG. 12. Representative images of the time lapse imaging can be seenfrom live cell imaging performed for the total period of 4 days whereinhibitor mix treated YFP⁺ multinucleated myotubes gave rise to YFP⁺mononucleated cells.

FIG. 13. FIG. 13A: Images that show ruling out any spurious YFPexpression in the absence of Cre expressing cells and in the presence ofinhibitor mix, 4 day old Lox YFP myotube cultures were treated with theinhibitor mix and then switched to growth medium. FIGS. 13B and 13C:Images that show no YFP⁺ mononucleated cells were observed in thecultures in spite of altered morphology of myotubes.

FIG. 14A-D. Tet-Oct4 myoblasts were infected with Ad-Cre and co-culturedwith Lox-YFP MB in DM for 96 hours, where myotubes were readily formed.

FIG. 15A-E. To further assess the properties of reprogrammed cells,mono-nucleated YFP⁺ progeny of de-differentiated myotubes was FACSsorted and expanded in culture. These YFP⁺ mononucleated cells wereimmunostained with antibody against Ki67 (proliferation marker) and BrdU(S phase marker) along with YFP.

DETAILED DESCRIPTION

Method for reprogramming differentiated cells into lineage restrictedprogenitor cells is provided. The method may include contactingdifferentiated cells with inhibitors of tyrosine phosphatases andapoptosis to de-differentiate differentiated cells into lineagerestricted progenitor cells, where the differentiated cell and thelineage restricted progenitor cells have the same lineage.

Before the present methods, reagents and kits are described further, itis to be understood that this invention is not limited to particularmethods, reagents and kits described, as such may, of course, vary. Itis also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupercedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the muscletissue” includes reference to one or more muscle tissues and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

The phrase “differentiated cell” as used herein refers to a post-mitoticcell of a cell lineage that has differentiated into a mature, functionalcell of a tissue. A differentiated cell expresses markers that arewell-known to the artisan as characteristic of a mature cell fate. Inaddition, because differentiated cells are post-mitotic, they do notincorporate BrdU into their DNA or express markers that are typicallyexpressed in proliferating cells, e.g. Ki67, PCNA, Anillin, AuroraB,Survivin, and the like. An example of a differentiated cell is ahepatocyte, a neuron, a myocyte, such as, a cardiomyocyte, a myofiber,and the like.

The term “dedifferentiation” or “dedifferentiates” as used herein refersto the process of reprogramming of a differentiated cell into a lessdifferentiated state than the differentiated cell in the same celllineage. In other words, when a differentiated cell dedifferentiates,the cell loses traits, e.g., morphology, expression of certain genes,functional capabilities, etc. of the differentiated cell and acquiretraits of cells of the lineage that are less mature.

The phrase “lineage-restricted progenitor cells” as used herein refersto cells having a defined lineage and that divide to produce cellshaving the same lineage. In other words, a lineage-restricted progenitorcell has committed to a certain lineage and hence is not a pluripotentcell that can produce different cell types. Rather, a lineage-restrictedprogenitor cell divides to produce cells of the same lineage as thelineage-restricted progenitor cell. Lineage-restricted progenitor cellsare identifiable by certain markers, such as, expression of one or moremarker proteins that are known in the art to be characteristic of aprogenitor cell for their cell lineage. In addition, progenitor cellsare typically mitotic, and thus incorporate BrdU into their DNA and/orexpress one or more markers, e.g. proteins that are typically expressedin mitotic cells, e.g. Ki67, PCNA, Anillin, AuroraB, and Survivin. Anexample of lineage-restricted progenitor cell is a progenitor cell ofthe muscle lineage, i.e., a myogenic progenitor cell, namely a myoblast,as it can give rise to more myoblasts and/or post-mitotic muscleprecursors that differentiate to produce myotubes.

The phrase “myogenic progenitor cells” as used herein refers to cellsthat divide to produce more myogenic progenitor cells that are capableof differentiating into post-mitotic muscle precursors and/or myotubes.A myogenic progenitor cell may be a myoblast. A myogenic progenitor cellis identifiable by a mononucleated morphology, and/or presence ofproliferation marker, such as, Ki67, and/or BrdU incorporation, and/orexpression of myogenic markers such as, MyoD1, Pax7, and the like.

The phrase “tyrosine phosphatase” as used herein refers to an enzymethat removes phosphate group from the tyrosine amino acid of asubstrate, such as, a protein substrate. Tyrosine phosphatases includephosphatases that include the active site called the CX5R motif.

The term “proliferate” as used herein refers to division of cells bymitosis, i.e., cells undergoing mitosis.

The phrase “expanded population” as used herein refers to a populationof cells that has proliferated, i.e., undergone mitosis, such that theexpanded population has an increase in cell number, that is, a greaternumber of cells, than the population at the outset.

The term “explant” refers to a portion of an organ or tissue taken fromthe body of a subject and cultured in an artificial medium. Cells thatare grown “ex vivo” are cells that are taken from the body in thismanner, temporarily cultured in vitro, and returned to the body.

The term “primary culture” denotes a mixed cell population of cells froman organ or tissue. The word “primary” takes its usual meaning in theart of tissue culture.

The terms “individual,” “subject,” “host,” and “patient,” are usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseaseor symptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom.

An “isolated” cell is one which has been separated and/or recovered froma component of the environment in which it was produced. Contaminantcomponents of its production environment are materials which wouldinterfere with culturing, screening, diagnostic or therapeutic uses forthe cell, and may include, other cell types, such as, neurons, proteins,enzymes, and other proteinaceous or nonproteinaceous components.

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998), the disclosures of which areincorporated herein by reference. Reagents, cloning vectors, and kitsfor genetic manipulation referred to in this disclosure are availablefrom commercial vendors such as BioRad, Stratagene, Invitrogen,Sigma-Aldrich, and ClonTech.

Method for Producing Lineage-Restricted Progenitor Cells

The methods of the present disclosure are based on the discovery thatcontacting a differentiated cell with an agent that inhibits tyrosinephosphatases and an agent that inhibits apoptosis results indedifferentiation of the cell and production of progenitor cells thatshare the same lineage as the differentiated cell. The use of an agentthat inhibits tyrosine phosphatases and an agent that inhibits apoptosisfor contacting a differentiated cell provides for a synergistic effectthat significantly increases the number of lineage restricted progenitorcells produced than that obtained by using either agent individually.Moreover, this synergistic effect is unexpected because either agentwhen used individually did not result in production of lineagerestricted progenitor cells from a differentiated cell to a significantdegree.

Accordingly, a method for generating lineage-restricted progenitor cellsfrom a differentiated cell is provided. In certain embodiments, themethod may include contacting a differentiated cell with an effectiveamount of an agent that inhibits tyrosine phosphatases and an effectiveamount of an agent that inhibits apoptosis under conditions sufficientfor generation of lineage-restricted progenitor cells from thedifferentiated cell, wherein the differentiated cell and thelineage-restricted progenitor cells have the same lineage.

In certain cases, the contacting may result in division of thedifferentiated cell to produce progeny, which progeny may includelineage-restricted progenitor cells. In certain embodiments,dedifferentiation of the differentiated cell may precede division.

In some embodiments, subject differentiated cells are contacted with theagents ex vivo, that is, the differentiated cells are harvested from thebody of a subject and contacted with the agents in vitro. In cases whenthe method is to be performed ex vivo, the differentiated cells may becultured from an explant, e.g. biopsy or autopsy material, as a cultureof primary cells. Methods of culturing differentiated cells fromexplants are typically specific for the type of primary cell beingcultured, and are well known to one of ordinary skill in the art. Forexample, cardiomyocytes may be isolated and cultured as described inMitcheson, J S et al. (1998) Cardiovascular Research 39(2):280-300. Anexemplary method for isolation and culturing of human skeletal musclemyocytes is provided in Rosenblatt et al. (1995) In Vitro Cell Dev. BiolAnim 31(10):773-339 (for human skeletal muscle myocytes). An exemplarymethod for isolation and culturing of intestinal smooth muscle myocytescan be found in Graham M, and Willey A. (2003). Methods in MolecularMedicine: Wound healing 78:417-423 Siow, R C M and Pearson, J D (2001)Methods in Molecular Medicine Angiogenesis protocols 46:237-245 providemethods for culturing of isolated vascular smooth muscle myocytes.

The differentiated cells are contacted ex vivo or in vivo with aneffective amount of an agent that inhibits tyrosine phosphatases and aneffective amount of an agent that inhibits apoptosis. As discussedherein, an agent that inhibits activity of tyrosine phosphatases is anagent that transiently antagonizes, inhibits or otherwise negativelyregulates the activity of tyrosine phosphatases that are upregulatedduring maturation of a progenitor cell into a differentiated cell;agents that inhibit activity of tyrosine phosphatases can therefore actanywhere along a tyrosine phosphatase signaling pathway as it is knownin the art. In certain embodiments, the inhibitor of tyrosinephosphatase activity acts directly on the tyosne phosphatases to inhibitits activity rather than a protein downstream to the tyrosinephosphatase in a signaling pathway. In certain cases, the agent may bean irreversible inhibitor of the tyrosine phosphatases. In other cases,the agent may be a transient or reversible inhibitor of the tyrosinephosphatases. Similarly, an agent that inhibits apoptosis is an agentthat transiently or irreversibly antagonizes, inhibits or otherwisenegatively regulates apoptosis. Agents that find use in the subjectmethod of generating lineage-restricted progenitor cells from adifferentiated cell are further described below.

An effective amount of an agent that inhibits tyrosine phosphatases isan amount that will reduce the overall activity of the tyrosinephosphatases or the downstream signaling pathway(s) in a differentiatedcell by at least about 25%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, by about 100%, such that the cellis able to enter mitosis and divide. Put another way, of the tyrosinephosphatases or the downstream signaling pathway(s) in a differentiatedcell may be reduced by at least about 2-fold, usually by at least about5-fold, e.g., 10-fold, 15-fold, 20-fold, 50-fold, 100-fold or more, ascompared to a control, such as, a differentiated cell not contacted bythe agent. For agents that inhibit the activity of tyrosine phosphatasesex vivo or in vitro, this effective amount may be measured by assayingdephosphorylation of substrates of the tyrosine phosphatases.

An effective amount of an agent that inhibits apoptosis is an amountthat will reduce apoptosis in the differentiated cell or adedifferentiated cell or a lineage restricted progenitor cell by atleast about 25%, at least 50%, at least 60%, at least 70%, at least 80%,at least 90%, at least 95%, by about 100%, such that the differentiatedcell is able to enter mitosis and divide to produce lineage-restrictedprogenitor cells. The effective amount of an inhibitor of apoptosis foruse in vitro or ex vivo may be determined by assaying apoptosis in inthe differentiated cell or a dedifferentiated cell or a lineagerestricted progenitor cell. Apoptosis may be assayed as known in theart.

By transiently, it is meant that the inhibition is for a limited periodof time, such as, for about 3 hours, about 6 hours, about 12 hours,about 1 day, about 2 days, about 3 days, about 5 days, about 7 days,about 10 days, about 15 days, about 20 days, or about 30 days.

The contacting of the differentiated cell with an effective amount of anagent that inhibits tyrosine phosphatases and an effective amount of anagent that inhibits apoptosis may be simultaneous or sequential. Forexample, the agent(s) that inhibits the activity of tyrosinephosphatases may be provided first, and the agent(s) that inhibitsapoptosis may be provided second, or vice versa, e.g., 1 hour later, 3hours later, 6 hours later, 12 hours later, 18 hours later, or 24 hourslater, or even later.

In some embodiments, additional agents that promote mitosis may beprovided to the cell at the contacting step, e.g. growth factors, e.g.bFGF, EGF, BMP, neuregulin, periostin; bovine groth serum or humangrowth serum, etc. In some embodiments, agents that promote cell cyclereentry are also provided to the cell in the contacting step. Forexample, in embodiments in which the subject differentiated cell is askeletal muscle myocyte, agents that disrupt microtubules such amyoseverin peptide (Rosania G R et al. (2000) Nat. Biotechnol.18(3):304-8) may be provided to fragment the multinucleated skeletalmuscle cell. Such agents are typically used when the subjectpost-mitotic differentiated cell has a morphologically complexphenotype, for example, a cytoskeletal architecture that polarizes thecell, such as the architecture of a multinucleated muscle cell, neuron,hepatocyte, etc.

In some embodiments, the contacting step may be carried out in absenceof growth factors, where the growth factors may be provided later, suchas, 12 hours, 18 hours, 24 hours, 36 hours, 72 hours, or later aftercontacting the differentiated cell with an agents that inhibits tyrosinephosphatases and an agent that inhibits apoptosis. The agents may beremoved after a certain period of time, i.e., the contacting step may becarried out for 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 6 hours, 8hours, 12 hours, 18 hours, 24 hours, 36 hours, 72 hours, or longer.During the contacting step the culture medium may be replaced with freshmedium containing the agents.

In embodiments in which the differentiated cells are induced to becomelineage-restricted progenitor cells and divide in vivo, i.e., in situ,agents may be administered locally, that is, directly to the target sitein a subject, i.e., the tissue, such as, a muscle tissue where treatmentis needed. The agents may be provided in any number of ways that areknown in the art, e.g., as a liquid (e.g. in any suitable buffer(saline, PBS, DMEM, Iscove's media, etc.)), as a paste, in a matrixsupport, conjugated to a solid support (e.g. a bead, a filter such as amesh filter, a membrane, a thread, etc.), etc. The conditions in thetissue are typically permissive of dedifferentiation and division oflineage-restricted progenitor cells, and no alteration of the basalconditions is required with the exception of providing the agents asdescribed above.

Inhibition of tyrosine phosphatases and apoptosis will induce thedifferentiated cell to become lineage-restricted progenitor cell anddivide to produce more lineage-restricted progenitor cells. Thelineage-restricted progenitor cell may undergo mitosis for a limitedamount of time, i.e., 12 hours, 1 day, 2 days, 3 days, 5 days, 7 days,10 days, 15 days, or 20 days. Accordingly, the lineage-restrictedprogenitor cells produced by the methods of the present disclosure mayundergo 1 round of mitosis, up to 2 rounds of mitosis, up to 3 rounds,up to 4 rounds, up to 5 rounds, up to 6 rounds, up to 10 rounds, up to30 rounds, up to 40 rounds, up to 50 rounds, or up to 60 rounds mitosis.As such, the lineage-restricted progenitor cells produced by the subjectmethods are unlike tumorigenic cells, which undergo unregulated mitosis,i.e., continue to divide for an unlimited amount of time. The period oftime in which the lineage-restricted progenitor cells are activelydividing is known as the induction period. During the induction period,a lineage-restricted progenitor cells that is induced to divide willgive rise to a population, or cohort, of progeny that arelineage-restricted cells. In other words, a lineage-restrictedprogenitor cells may give rise to 2 or more cells, 4 or more cells, 8 ormore cells, 16 or more cells, 32 or more cells, 64 or more cells, 100 ormore cells, 1000 or more cells, or 10,000 or more cells. In someembodiments, at least about 1%, about 2%, about 5%, about 8%, moreusually about 10%, about 15%, about 20%, or about 50% of contacteddifferentiated cells in a population may be induced to dedifferentiateand divide.

Following production of lineage-restricted progenitor cells, these cellsmay be subject to conditions that induce the cells to differentiate toproduce differentiated cell of the same lineage as that of thelineage-restricted progenitor cells and the differentiated cell fromwhich the lineage-restricted progenitor cells were produced. In otherembodiments, the lineage-restricted progenitor cells may spontaneouslydifferentiate into differentiated cells.

In certain embodiments, the transferring of the cells induced to divideex vivo to condition that promote differentiation is effected bytransplanting the progeny into the tissue of a subject. Cells may betransplanted by any of a number of standard methods in the art fordelivering cells to tissue, e.g. injecting them as a suspension in asuitable buffer (saline, PBS, DMEM, Iscove's media, etc.), providingthem on a solid support, e.g. a bead, a filter such as a mesh filter, amembrane, etc. In certain embodiments, the differentiation may becarried out by changing the culture medium to a medium that promotes thedifferentiation of cells of that lineage, as is known in the art.

In general, the subject methods achieve the dedifferentiation of adifferentiated cell without the use of exogenous gene expression tomodulate the expression of gene(s) involved in maintainingdifferentiated cell in a differentiated state or genes mediatingreversal of a differentiated cell into a dedifferentiate state.

Differentiated Cell

A variety of differentiated cells may be used in the methods provided inthe present disclosure. As noted above, a differentiated cell is a cellthat has completed differentiation to become mature functional cell.Examples of differentiated cells include a myocyte in skeletal or heartmuscle, an islet cell in pancreas, a hepatocyte in liver, a neuron incentral nervous system, a neuron in peripheral nervous system, anosteocyte in bone, hematopoietic cell from blood, and the like. Adifferentiated cell can be identified as such by the expression of oneor more proteins or RNAs, i.e. markers for the type of differentiatedcell, as known in the art.

In some embodiments, the subject differentiated cells are myocytes,which express one or more of myogenin, myosin heavy chain (MHC), andcreatine kinase. In certain embodiments, the myocytes arecardiomyocytes, which are rod shaped and cross-striated in culture andexpress one or more of proteins cardiac troponin, eHand transcriptionfactor, and cardiac-specific myosins. In certain embodiments, themyocytes are smooth muscle myocytes, which express smooth muscle actin.In certain embodiments, the myocytes are skeletal muscle myocytes, whichexpress one or more of skeletal muscle myosins, skeletal muscletroponin, myoD.

In certain embodiments, the differentiated cell may be a myocyte and thecontacting may result is division of the myocyte into myogenicprogenitor cells. In certain embodiments, the myocyte may be selectedfrom the group consisting of cardiomyoctyte, a smooth muscle myocyte,and a skeletal myocyte.

A skeletal muscle myocyte may be identified by expression of RNA orproteins, such as, eMyHC, myogenin, p21, p15, and p16 expression and/orby cell morphology, such as, shape, presence of multiple nuclei in asingle cell, and/or lack of cell proliferation.

In certain embodiments, the differentiated cell used in the methodsprovided in the present disclosure may be isolated from a subject or anindividual, such as, a patient needing tissue regeneration. For example,the differentiated cell may be isolated from a tissue sample obtainedfrom a subject. In certain examples, the tissue sample may be a biopsysample, such as, a biopsy sample of a muscle of the subject. In certainembodiments, the muscle tissue may be removed from the heart, bloodvessel, intestine, or a limb of the subject. The differentiated cell maybe isolated from the biopsy sample by methods known in the art. Anexemplary method for isolation of myocytes from muscle tissue isdescribed in (Conboy and Conboy, Methods Mol Biol 621, 149-163, 2010).

Differentiated cells useful for producing lineage-restricted cellsinclude any post-mitotic mature cell from any tissue comprisingpost-mitotic mature differentiated cells, e.g., muscle, nervous system,pancreas, liver, etc., e.g., a cardiomyocyte from an individual with aheart condition, a myocyte from an individual with muscular dystrophy, aneuron from an individual with Alzheimer's disease, Parkinson's Disease,ALS, and the like, as described above.

Inhibitors

As noted above, the method includes contacting a differentiated cellwith an agent that inhibits tyrosine phosphatases and an agent thatinhibits apoptosis.

In certain embodiments, the agent that inhibits tyrosine phosphatasesand the agent that inhibits apoptosis may be is a small molecule that iscell permeable.

A small molecule compound may range in molecular weight from 50 daltonsto 2500 daltons, such as, 100 daltons to 2000 daltons, or 200 daltons to1000 daltons, such as 100 daltons, 300 daltons, 400 daltons, 500daltons, 800 daltons, 1000 daltons, 1500 daltons, or 2500 daltons.

Naturally occurring or synthetic small molecules of interest includenumerous chemical classes, such as organic molecules, e.g., smallorganic compounds having a molecular weight of more than 50 daltons andless than about 2,500 daltons. An agent used in the methods disclosedherein may include functional group(s) for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group. The agents may includecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups.

Small molecule inhibitors of tyrosine phosphatases and apoptosis can beprovided directly to the medium in which the cells are being cultured,for example, as a solution in DMSO, water, or other solvent.

In certain embodiments, the agent that inhibits tyrosine phosphatasesmay include a small molecule inhibitor of activity of tyrosinephosphatases. In general, the agent inhibits the activity of two or moretyrosine phosphatases, such as, at least two, three, four, or moretyrosine phosphatases.

In certain cases an agent that inhibits tyrosine phosphatases may be avandate small molecule, peroxovanadium (pV) small molecule, or abisperoxovanadium (bpV), or derivatives thereof. In certain embodiments,an agent that inhibits tyrosine phosphatases may include one or more ofpotassium bisperoxo (bipyridine) oxovanadate (bpV(bipy), potassiumbisperoxo(1,10-phenanthroline)oxovanadate (pV(phenanthroline) orbpV(phen)), potassium bisperoxo (piconlinate) oxovanadate (pV(pic)) andpotassium bisperoxo(phenylbiguanide) oxovanadate (pV(biguan)). Incertain embodiments, one or more of the vanadium compounds such as thosedescribed in U.S. Pat. No. 7,692,012 may be used as an agent to inhibittyrosine phosphatases.

In certain cases, the agent that inhibits tyrosine phosphatases may bean agent that is an inhibitor of protein tyrosine phosphatases (PTPs),such as, Class I PTPs that include receptor tyrosine phosphatases,non-receptor type PTPs, dual-specific phosphatases, such as, MAPKphosphatases, slingshots, phosphatase and tensin homologs (PTENs); ClassII PTPs that include low molecular weight phosphotyrosine phosphatase;Class III PTPs that include Cdc25 phosphatases; and Class IV PTPs thatinclude pTyr-specific phosphatases.

In certain cases, an agent that inhibits apoptosis may include an agentthat inhibits one or more proteins that mediate apoptosis. In someexamples, the agent may include one or more agents that inhibit one ormore of a caspase, a calpain, and/or Bcl-xL. In certain cases, the agentmay inhibit the function of one or more caspases. In some embodiments,the inhibitor may inhibit one or more of the caspases, such as, caspase1, caspase 2, caspase 3, caspase 8, caspase 9, caspasel 0, and/orcaspase 12.

In certain cases, an apoptosis inhibitor may be an agent that forms anirreversible thioether bond between the aspartic acid derivative in theinhibitor and the active site cysteine of the caspase with thedisplacement or the 2,6-difluorophenoxy leaving group. In certain cases,an agent that inhibits apoptosis may includeN-(2-Quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methyl ketone which isa cell permeable, irreversible pan-caspase inhibitor, especially activeagainst caspases 1, 3, 8, and 9. The compound is also referred to as“Q-VD-oPh” or “OPH-109” or Q-VD-OPH. The compound has a quinolinederivative (Q), a dipeptide, valine (V, in standard single letter code)and aspartic acid (D, in standard single letter code), and a non toxic2,6-difluorophenoxy methylketone (OPH) group.

In certain embodiments, one or more agents that inhibit apoptosis may beused in the methods disclosed herein.

In certain cases, an agent that inhibits apoptosis may be a broadspectrum caspase inhibitor such as Q-VD-OPH, Z-VAD-FMK (ZVAD-fmk) orBOC-D-FMK (Boc-D-fmk).

One or more agents that inhibit the activity of tyrosine phosphatasesmay be used. Likewise, one or more agents that inhibit the apoptosis maybe used. The agents may be provided to the differentiated cellsindividually or as a single composition, that is, as a premixedcomposition, of agents. When provided individually, the agents may beadded to the subject differentiated cells simultaneously or sequentiallyat different times.

In certain cases, the agent that inhibits tyrosine phosphatases may be abroad spectrum inhibitor, i.e., it may inhibit the activity of two ormore tyrosine phosphatases involved in promoting and/or maintaindifferentiated state of a differentiated cell, such as, three, or four,or five, or more tyrosine phosphatases.

In Vivo Use of Lineage Restricted Progenitor Cells

The progeny of the dedifferentiated cell, i.e., lineage-restrictedprogenitor cells may be used for supplying differentiating ordifferentiated cells to a recipient for regenerating tissue. Forexample, in embodiments of the above methods in which the differentiatedcells are myocytes, transplanting lineage-restricted cells generated themethods described above into muscle, or producing lineage-specific cellsin situ in the muscle by in vivo methods described above results in thegeneration of new muscle cells in the patient. Muscle regeneration asused herein refers to the process by which new muscle fibers form frommyogenic progenitor cells or muscle precursor cells. Thelineage-restricted progenitor cells produced by the subject methods maybe administered to a patient in a composition. The composition willusually confer an increase in the number of new fibers by at least 1%,more preferably by at least 20%, and most preferably by at least 50%.The growth of muscle may occur by the increase in the fiber size and/orby increasing the number of fibers. The growth of muscle may be measuredby an increase in wet weight, an increase in protein content, anincrease in the number of muscle fibers, an increase in muscle fiberdiameter, etc. An increase in growth of a muscle fiber can be defined asan increase in the diameter where the diameter is defined as the minoraxis of ellipsis of the cross section. Productive muscle regenerationmay be also monitored by an increase in muscle strength and/or agility.

Tissue regeneration therapy that employs the lineage-restricted cellsproduced by the subject methods are useful for treating subjectssuffering from a wide range of diseases or disorders. For example, inembodiments in which the post-mitotic differentiated cells are myocytes,subjects suffering from muscular disorders, e.g., acute cardiacischemia, injury due to surgery (e.g. tumor resection) or physicaltrauma (amputation/gunshot wound), or degenerative heart diseases suchas ischemic cardiomyopathy, conduction disease, and congenital defects,etc. could especially benefit from regenerative tissue therapies thatuse the lineage-restricted cells of the subject method.

Other examples of muscle disorders that could be treated with thesubject cells, such as, allogeneic cells, autologous cells, and/orgenetically modified autologous cells, include muscular dystrophies suchas Duchenne dystrophy and Becker muscular dystrophy.

Other particular examples of muscle disorders that could be treated withthe subject cells, either allogeneic cells, autologous cells, and/orgenetically modified autologous cells, include the non-dystrophicmyopathies such as congenital and metabolic myopathies, includingglycogen storage diseases and mitochondrial myopathies, channelopathies,myotonic disorders, myotonic dystrophy (Steinert's disease), myotoniacongenita (Thomsen's disease).

Particular examples of muscle disorders that could be treated with thesubject cells include disorders of the heart muscle. Such disordersinclude, without limitation, myocardial infarction (interruption ofblood supply to a part of the heart, causing heart cells to die);cardiac arrest (failure of the heart to contract effectively); heartfailure (a progressive inability of the heart to supply sufficient bloodflow to meet the body's needs, often but not always due to myocardialinfarction or cardiac arrest); cardiac ischemia reperfusion injury(injury to a tissue due to reperfusion of the tissue with bloodfollowing an ischemic condition); cardiomyopathy (muscle weakness due toe.g. ischemia, drug or alcohol toxicity, certain infections (includingHepatitis C), and various genetic and idiopathic (i.e., unknown)causes); injury due to surgery, and degenerative heart diseases such asconduction disease and congenital defects.

In certain cases, the lineage restricted progenitor cells may be used tosupplement the number of differentiated cell in a subject in whom therehas been a decline in the number of differentiated cell due to aging,such as, a decrease in muscle mass, neurons, and the like.

Diseases other than those of the musculature may similarly be treated byregenerative tissue therapy that employs lineage-restricted cellsproduced by the subject methods. For example, diseases of the centralnervous system (CNS) or the peripheral nervous system (PNS) may betreated by such therapy. For example, for the treatment of Parkinson'sdisease, dopaminergic neurons may be transiently induced to divide,giving rise to neural progenitors (i.e. mitotic cells of the neurallineage) or neural precursors (post-mitotic cells of the neural lineage,i.e. following exit from mitosis) that may be transferred into thesubstantia nigra of a subject suffering from Parkinson's disease.Alternatively, the neural progenitors or neural precursors may beinduced to differentiate into dopaminergic neurons ex vivo, and thentransferred into the substantia nigra or striatum of a subject sufferingfrom Parkinson's disease. Alternatively, dopaminergic neurons of thesubstantia nigra of a subject suffering from Parkinson's disease may beinduced to transiently divide in situ. Descriptions of post-mitoticdifferentiated neurons, neuronal progenitor and precursor cells, andmethods for culturing these cells are have been described in the art.Other diseases and disorders of the nervous system that may benefit fromthe subject methods include Alzheimer's Disease, ALS, disorders ofolfactory neurons, a disorder of spinal cord neurons, a disorder ofperipheral neurons, and other disorders due to injury or disease.

For the treatment of multiple sclerosis, spinal cord injury, or otherdisorder of the central nervous system in which enhancing myelination isdesirable to treat the disorder, oligodendrocytes may be induced todivide, giving rise to oligodendrocyte progenitors or oligodendrocyteprecursors, which are then transferred to a subject suffering from ademyelinating condition of the CNS, e.g. multiple sclerosis or anothercondition where it is desirable to enhance myelination, e.g., spinalcord injury, etc. The lineage restricted progenitor cells may betransplanted at the site where enhanced myelination is desired.Alternatively, the oligodendrocyte progenitors or oligodendrocyteprecursors may be induced to differentiate into oligodendrocytes exvivo, and then transferred into the subject suffering from the MS,spinal cord injury, etc., at the site where enhanced myelination isdesired. Alternatively, oligodendrocytes of a subject suffering from theMS, spinal cord injury, etc. may be induced to transiently divide insitu at the site where enhanced myelination is desired. Descriptions ofpost-mitotic differentiated oligodendrocytes, oligodendrocyteprogenitors, and oligodendrocyte precursors, and how to culture thesecells are described in Dugas, J. et al. (2006) J. Neurosci.26:10967-10983 and US Application No. 20090258423.

In other examples, pancreatic islet cell progenitor or precursor cellsgenerated from post-mitotic differentiated pancreatic islet cells may betransplanted into a subject suffering from diabetes (e.g., diabetesmellitus, type 1), see e.g., Burns et al., (2006) Curr. Stem Cell Res.Ther., 2:255-266. Descriptions of post-mitotic differentiated cells ofthe pancreas, i.e., islet cells, the progenitor and precursor cells ofthat lineage, and methods for culturing these cells are described inU.S. Pat. No. 6,326,201, the disclosure of which is incorporated hereinby reference.

Hepatic progenitor cells or post-mitotic differentiated hepatic cellsderived from post-mitotic differentiated hepatic cells are transplantedinto a subject suffering from a liver disease, e.g., hepatitis,cirrhosis, or liver failure.

In some instances, it will be desirable to regenerate tissue withlineage-restricted cells that were produced from post-mitoticdifferentiated cells of allogeneic tissue, that is, tissue from adifferent host, for example, where the disease conditions result fromgenetic defects in tissue-specific cell function. Where the dysfunctionarises from conditions such as trauma, the subject cells may be isolatedfrom autologous tissue, and used to regenerate function. Autologouscells may also be genetically modified, in order to correct diseaseconditions resulting from genetic defects. Alternatively, where thedysfunction arises from conditions such as trauma, post-mitoticdifferentiated cells may be transiently induced to divide in situ,giving rise to lineage-restricted cells that will differentiate andincorporate into the injured tissue.

As alluded to above, genes may be introduced into the subjectlineage-restricted cells that have been produced ex vivo for a varietyof purposes, e.g. to replace genes having a loss of function mutation,provide marker genes, etc. Alternatively, vectors are introduced thatexpress antisense mRNA or ribozymes, thereby blocking expression of anundesired gene. Other methods of gene therapy are the introduction ofdrug resistance genes to enable normal progenitor cells to have anadvantage and be subject to selective pressure, for example the multipledrug resistance gene (MDR), or anti-apoptosis genes, such as bcl-2.Various techniques known in the art may be used to introduce nucleicacids into the lineage-restricted cells, e.g. electroporation, calciumprecipitated DNA, fusion, transfection, lipofection, infection and thelike, as discussed above. The particular manner in which the DNA isintroduced is not critical to the practice of the subject methods.

In Vitro Uses of Lineage-Restricted Progenitor Cells

Lineage-restricted progenitor cells may be used for generating celllines that may be used for characterization of a disease. For example,differentiated cells may be obtained from a subject suffering from adisease that has not yet been well characterized. These cells may beused to generate lineage-restricted progenitor cells that also exhibitthe disease phenotype. Thus, the propagated lineage-restrictedprogenitor cells may serve the characterization of the regulatorymechanisms that have been perturbed in the disease. In addition, thesecells will serve as material on which to screen therapeutic agents fortheir ability to ameliorate the disease phenotype.

In screening assays for biologically active agents, lineage-restrictedprogenitor cells are produced from differentiated cells from anindividual, e.g., an individual with a disease condition, e.g., a liveindividual or a cadaver, by the subject methods described above, andallowed to differentiate. The differentiated cells are then contactedwith a candidate agent of interest and the effect of the candidate agentis assessed by monitoring amelioration of one or more phenotype of thedisease.

Screening Methods

Methods for screening for an agent that inhibits tyrosine phosphatasesand an agent that inhibits apoptosis and mediates generation oflineage-restricted progenitor cells from a differentiated cell are alsoprovided. The method may include contacting a differentiated cell with acandidate agent that inhibits tyrosine phosphatases and a candidateagent that inhibits apoptosis under conditions sufficient for generationof lineage-restricted progenitor cells from the differentiated cell,wherein the differentiated cell and the have the same lineage; anddetermining whether lineage-restricted progenitor cells are produced,wherein the presence of lineage-restricted progenitor cells indicatesthat the candidate agent that inhibits tyrosine phosphatases and thecandidate agent that inhibits apoptosis mediate generation oflineage-restricted progenitor cells from a differentiated cell.

Candidate agents of interest for screening include biologically activeagents of numerous chemical classes, primarily organic molecules,although including in some instances, inorganic molecules,organometallic molecules, immunoglobulins, genetic sequences, etc. Alsoof interest are small organic molecules, which comprise functionalgroups necessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, frequently at least two of the functionalchemical groups. The candidate agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Candidateagents are also found among biomolecules, including peptides,polynucleotides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.

Compounds may be obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds, including biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

A plurality of assays may be run in parallel with differentconcentrations to obtain a differential response to the variousconcentrations. As known in the art, determining the effectiveconcentration of an agent typically uses a range of concentrationsresulting from 1:10, or other log scale, dilutions. The concentrationsmay be further refined with a second series of dilutions, if necessary.Typically, one of these concentrations serves as a negative control,i.e., at zero concentration or below the level of detection of the agentor at or below the concentration of agent that does not give adetectable change in dedifferentiation and production oflineage-restricted progenitor cells or decrease in activity of tyrosinephosphatases or inhibition of caspases.

In certain cases, the differentiated cell is as provided above. Incertain cases, the differentiated cell may be a myocyte and thelineage-restricted progenitor cells may be myogenic progenitor cells.

Kits

A kit for use in generation of lineage-restricted progenitor cells froma differentiated cell is also provided. The kit may include an agentthat inhibits tyrosine phosphatases and an agent that inhibitsapoptosis. The agents might be premixed in a single container orprovided in separate containers.

In certain embodiments, the kit may include an inhibitor of tyrosinephosphatase and an inhibitor of caspases as described herein. In certaincases, the kit may include a vanadium containing small molecule and angeneral caspase inhibitor, such as, those described herein.

EXAMPLE Materials and Methods

Animal Strains:

B6;129-Gt(ROSA)26Sor^(tm1(rtTA*M2)Jae) Col1a1^(tm2(tetOPou5f1)Jae)/Jstrain (Stock Number: 006911), B6.129X1-Gt(ROSA)26Sor^(tm1(EYFP)Cos)/Jstrain (Stock Number: 006148), NOD.CB17-Prkdcscid/J (Stock Number:001303) and C57BL6/J (2-3 months old) mice were obtained from pathogenfree breeding colonies at The Jackson Laboratories. Animals were housedat the Northwest Animal Facility, University of California, Berkeley andprocedures were performed in accordance to administrative panel on theOffice of Laboratory Animal Care, UC Berkeley.

Reagents, Antibodies, Western Blotting and Immunofluorescence:

BpV(phen) (Alexis Biochemicals), Doxycycline (Sigma) and apoptosisinhibitor (Q-VD-OPh, Non O methylated Cat No. 551476, Calbiochem), BrdU(Sigma), ECM (Sigma), Hoechst 33342 (Sigma), DNase1 (Sigma), propidiumiodide (molecular probes), Rnase A (Fermentas) and mouse bFGF (R&D) werepurchased. Antibodies to BrdU (ab6326), GFP (ab6556 and ab13970), Ki67(abcam ab155800) and Oct4 (ab18976) were from Abcam. Antibodies againstActin (rabbit polyclonal) were from Sigma, Pax7 was from DSHB(Develpmental Studies Hybridoma Bank), eMyHC from DSHB and Upstate,mouse monoclonal antibodies against Myogenin, MyoD and p21 were fromSanta Cruz Biotechnology. For western blotting, the cells were lysed inRIPA buffer (50 mM Tris-Cl pH7.6, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.25%sodium deoxycholate, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM PMSF, 1mM EDTA, 1× Protease inhibitor, Sigma) and protein concentrationdetermined by Bradford Assay. 30 μg protein was run on precast 4-20%Gels (BioRad), then transferred to nitrocellulose membrane for 2 hoursand protein expression detected by BioRad Gel Doc/Chemi Doc imagingsystem and Quantity One Software. For immunofluorescence, cells werefixed with 4% PFA for 15 minutes at room temperature followed bypermeabilization with 0.25% Triton-X 100 for 12 minutes and blocked forone hour in blocking buffer (1% BGS+0.1% Na-Azide in 1×PBS) followed byprimary antibody incubation in blocking buffer for 2 hours or overnightand secondary antibody incubation for 1 hour in blocking buffer withAlexa fluorophore conjugated species specific secondary antibody(Invitrogen). For BrdU labeling, cells were pulsed with 10 μM BrdU foreither 2 or 24 hours, fixed with 4% PFA for 15 minutes, permeabilizedwith 0.25% Triton-X 100 for 12 minutes followed by DNase1 treatment (0.2units/μL) for 30 minutes at room temperature. For allimmunofluorescence, cells were mounted with mounting media containingDAPI (Prolong Gold Antifade, Invitrogen) to visualize nuclei in allimmunostaining experiments.

Primary Myoblast Isolation:

Primary myoblasts were obtained by isolation of satellite cells fromthese mice as described previously (Conboy and Conboy, Methods Mol Biol621, 149-163, 2010). TA and Gastrocnemius muscle of the differenttransgenic mice were injected with total 5 μg cardiotoxin (Sigma)dissolved in 1×PBS and muscle was dissected out after 3 day injury asdescribed (Conboy and Conboy, Methods Mol Biol 621, 149-163, 2010). Inbrief, muscle underwent enzymatic digestion at 37° C. in DMEM(Cellgro)+1% Penicillin/Streptomycin+250 units/ml Collagenase Type II(Sigma) solution for one and half hour on rocker with slight agitation.Bulk myofibers were purified by repeated rounds of trituration,sedimentation and washing to remove interstitial cells, tendons etc.Satellite cells were purified from these bulk myofibers by incubation in0.5 units/ml dispase at 37° C. for half an hour followed bysedimentation, washing, and fine mesh straining. The satellite cellswere then cultured on 1:500 ECM (Sigma) coated plates in myoblast growthmedium containing Ham's F-10 nutrient mixture (Gibco)+20% Bovine GrowthSerum (BGS; Hyclone)+9 ng/ml bFGF (R&D)+1% Penicillin/Streptomycin.Later, proliferating fusion competent myoblasts were pre-plated toremove fibroblasts from culture.

Adenovirus Infection and Primary Myotube Labeling:

Wild type MB or Tet-Oct4 MB obtained were infected with Ad-CMV-Cre orAd-RFP control virus (Vector Biolabs) for 4-6 hours, washed off andcultured in GM for 24 hours. Ad-Cre MB were then lifted off the plates,counted and co-cultured with Lox-YFP myoblasts in differentiationinducing medium DMEM (Cellgro)+2% horse serum (Sigma)+1%Penicillin/Streptomycin (BD Falcon) on 12 well ECM coated plates. The 96hour old myotubes were visualized for YFP expression, treated withdifferent experimental conditions and photographed everyday using Zeissepifluorescence microscope Axio Observer A.1 fitted with Zeiss EYFPfilter (BP 500/20 FT515 BP 535/30) at 10× and 20× objective.

De-Differentiation Assay:

WT myoblasts or Tet-Oct4 (3×10⁵) myoblasts upon 4-6 hour infection with300 MOI of Ad-Cre virus were washed with growth medium twice to get ridof any residual virus and incubated for another 24 hours in growthmedium at 37° C. in 5% CO₂ incubator. These myoblasts were then liftedoff from the plates by addition of 1×PBS, counted (3×10⁴ cells/well) andco-cultured with 7×10⁴ Lox-YFP myoblasts (obtained fromB6.129X1-Gt(ROSA)26Sor^(tm1(EYFP)Cos)/J mice strain) per well indifferentiation inducing medium DMEM (Cellgro)+2% horse serum+1%Penicillin/Streptomycin on 12 well (BD Falcon) on ECM (1:500) coated 12well plates. Later, 96 hour old YFP⁺ myotube cultures were treated withBpV (phen) (10 μM) along with apoptosis/caspase inhibitor (Q-VD-OPh) (10μM) daily for 2 days with and without doxycycline (2 μg/ml) indifferentiation medium. The inhibitors were then withdrawn and cellswere switched to growth medium containing bFGF (9 ng/ml). The treatedcells were fed fresh growth medium and bFGF everyday over a period oftime and the morphology of YFP expressing myotubes was routinelyvisualized by epifluorescence microscope using YFP filter andphotographed using 10× and 20× objectives. For live Hoechst staining, 4μM of Hoechst was added everyday to YFP labeled myotube cultures,incubated for 10 minutes at 37° C. in 5% CO₂ incubator, washed off toremove unlabeled Hoechst and photographed.

Calculation of Myotube Reprogramming Efficiency:

The labeled YFP⁺ myotubes per well in 4 day old Cre-Lox myotube cultureswere determined to be ˜2600. After inhibitor treatment, ˜350 YFP⁺mononucleated cells were found in culture. Hence reprogrammingefficiency was calculated as the percent of YFP⁺ mononucleated cells outof the total number of labeled YFP⁺ myotubes which was determined˜13.46%. Another method was based on the determination of Lox YFPmyonuclei in labeled myotubes. As an average number of myonuclei presentin each YFP⁺ myotube is ˜4 and for myotube labeling, Cre and Lox YFPmyoblasts were cultured in ratio of 1:2 hence Lox YFP myonuclei wereestimated to be ˜6940 [(2600×4)×2/3]. Therefore, reprogrammingefficiency calculated was the percent of YFP⁺ mononucleated cells out ofan estimated total number of Lox YFP myonuclei in YFP⁺ myotubes and wasdetermined ˜5.04% [(350/6940)×100] (see FIG. 11D).

Cell Transplantation:

1×10⁶ reprogrammed YFP⁺ mononucleated cells expanded in GM in culturewere re-suspended in medium containing Ham's F-10 with 2% BGS andinjected in 24 hours cardiotoxin pre injured Tibialis Anterior (TA)muscles of NOD-SCID mice (4 weeks old) obtained from Jackson lab.Control injections were performed with medium alone or Lox YFP myoblastsre-suspended in medium. After 2-3 weeks of cell injections, TA muscleswere dissected out and fixed in 4% paraformaldehyde (PFA) for minimum of2 hours and subsequently washed three times with 1×PBS for total of 45minutes. The muscles were then sequentially transferred to 2%, 5% and10% sucrose in PBS with slight agitation at room temperature for 30minutes-1 hour. Finally muscles were left overnight in 20% sucrose at 4°C. and frozen in liquid nitrogen cooled iso-pentane in OCT embeddingmedium. 10 um muscle sections were cut and mounted on superfrost slides.300 um serially spaced muscle sections from whole block were post-fixedwith 4% PFA for 10 minutes and permeabilized for 10 minutes with 0.25%Triton X-100 and blocked in goat serum. Primary antibodies against YFP(chicken polyclonal GFP, Abcam) and laminin (rat laminin from Sigma)were added overnight followed by 1 hour incubation in the respectivefluorochrome conjugated secondary antibodies (Goat anti Chicken 488 andGoat anti rat 546) from Molecular probes.

RNA Isolation and qRT-PCR:

RNA isolation was performed by RNAeasy Kit (Qiagen) according tomanufacture recommendations. For real time-qPCR, transcribed cDNA wasdiluted 1:5 for every sample and 1 μl of this diluted cDNA was used for25 μl PCR reaction containing 80 nM forward and reverse primers, 12.5 μlof 2× iQ Syber Green mix (Bio-Rad), ran on iQ5 cycler (Bio-Rad) and dataanalyzed by iQ5 optical system software. The values normalized againstinternal control GAPDH and plotted using ΔΔCt method. The primersagainst specific genes for qRT-PCR are: GAPDH-F5′-GGGAAGCCCATCACCATCT-3′ (SEQ ID NO: 1) and GAPDH-R5′-GCCTCACCCCATTTGATGTT-3′ (SEQ ID NO: 2); Myogenin-F5′-GACCCTACAGACGCCCACAA (SEQ ID NO: 3) andMyogenin-R-5′-CCGTGATGCTGTCCACGAT-3′ (SEQ ID NO: 4); MyoD-F5′-CGGCTCTCTCTGCTCCTTTG-3′ (SEQ ID NO: 5) andMyoD-R-5′-GAGTCGAAACACGGGTCATCA-3′ (SEQ ID NO: 6);Pax7-F-5′-CCCTCAGTGAGTTCGATTAGC-3′ (SEQ ID NO: 7) andPax7-R-5′-CCTTCCTCGTCGTCCTCTTTC-3′ (SEQ ID NO: 8);p21-F-5′GAACATCTCAGGGCCGAAAA-3′ (SEQ ID NO: 9) andp21-R-5′-TGCGCTTGGAGTGATAGAAATC-3′ (SEQ ID NO: 10);eMyHC-F-5′-AGAGGACGTGTATGCCATGA-3′ (SEQ ID NO: 11) andeMyHC-R-5′-TGGCCATGTCCTCAATCTTGT-3′ (SEQ ID NO: 12);Cre-recombinase-F-5′-GCCGGGTCAGAAAAAATGG (SEQ ID NO: 13) and Crerecombinase-R-5′-AGGGCGCGAGTTGATAGCT-3′ (SEQ ID NO: 14); eYFPF-5′-GCACGACTTCTTCAAGTCCGCCATGCC-3′ (SEQ ID NO: 15) and eYFP R 5′-GCGGATCTTGAAGTTCACCTTGATGCC-3′ (SEQ ID NO: 16). Primers for p15 and p16have been described elsewhere (Li et al., Nature 460, 1136-1139, 2009).For SA biosciences/Qiagen PCR arrays, 0.5 μg RNA was reverse transcribedusing SA biosciences RT kit and PCR arrays carried out according tomanufacture instructions. Web based PCR array data analysis tool of SAbiosciences was utilized for data analysis.

Flow Cytometry:

De-differentiated YFP⁺ mononucleated cells were lifted off the plates byaddition of PBS and re-suspended in PBS+5% BSA, filtered through 40 uMfilter (BD Falcon) to remove any aggregates and placed on ice. Cellsorting was performed on Cytopeia Influx sorter with gating on YFP⁺population set by a comparison with negative control sample of Lox-YFPmyoblasts. The sorted cells were replated on ECM coated tissue culturedishes containing myoblast growth medium with 9 ng/ml bFGF and assessedfor different markers. For cell cycle analysis, de-differentiated cellswere expanded in culture, pelleted down and fixed in ice cold 70%ethanol overnight at −20° C. Next day, cells were pelleted down at 1400rpm for 5 minutes at 4° C. and ethanol was removed. Cells were washedonce with ice cold 1×DPBS and re-suspended in DNA staining solution(RNase 0.1 mg/ml, Propidium iodide 2 μg/ml, 0.05% Triton X 100 in1×DPBS) for 30 minutes at room temperature. The cells were pelleteddown, washed once with ice cold DPBS and resuspended in DPBS+3% BSA forFACS analysis.

Statistical Analysis:

p values were determined using student's t-test (2 samples equalvariance, 2 tailed).

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Abbreviations

YFP, Yellow fluorescent protein; RFP, Red fluorescent protein, dox,doxycycline; DM, differentiation medium; GM, growth medium; BpV,bisperoxovanadium; bFGF, basic fibroblast growth factor; eMyHC,embryonic myosin heavy chain; GAPDH, glyceraldehyde phosphatedehydrogenase; MB, myoblasts; MT, myotubes.

Overview

Muscle regeneration declines with aging and myopathies, andreprogramming of differentiated muscle cells to their progenitors canserve as a robust source of therapeutic cells. Here, the Cre-Lox methodwas used to specifically label post-mitotic primary multinucleatedmyotubes and then small molecule inhibitors of tyrosine phosphatases andapoptosis were utilized to de-differentiate these myotubes intoproliferating myogenic cells, without gene over expression. Thereprogrammed, fusion competent, muscle precursor cells contributed tomuscle regeneration in vitro and in vivo and were unequivocallydistinguished from reactivated reserve cells due to the lineage markingmethod. The small molecule inhibitors down-regulated cell cycleinhibitors and chromatin remodeling factors known to promote andmaintain the cell fate of myotubes, facilitating cell fate reversal.These findings enhance understanding of cell-fate determination andcreate novel therapeutic approaches for improved muscle repair.

Example 1 Fusion-Dependent Lineage Marking of Primary Myotubes

To overcome instances of mistaken cell identity during reprogrammingstudies, a method to genetically and irreversibly label terminallydifferentiated myotubes using the Cre-Lox technique was firstestablished (FIG. 1A). The Cre-Lox system has been widely used fortissue specific disruption of genes, utilizing the P1 bacteriophage Crerecombinase which specifically recognizes Lox sites and excises any DNAsequence flanked by these sites (Nagy, 2000). The schematic of thesystem is represented in FIG. 1A. Cre recombinase was expressedexogenously by adenoviral mediated infection (Ad-Cre) in wild typeprimary myoblasts (MB). Ad-Cre infected myoblasts (Cre-MB) wereco-cultured with Lox-YFP myoblasts (Lox-YFP MB) derived from Rosa26 YFPreporter mice (Srinivas et al., BMC Dev Biol 1, 4, 2001) in the ratio of1:2 so that more number of Lox YFP myonuclei coexist with Ad-Cremyonuclei in single myotube and yield high expression of YFP. Instandard low mitogen differentiation-promoting medium (DMEM, 2% horseserum), these co-cultured MB within 72-96 hours physiologically fusedinto YFP expressing myotubes (hence forth, YFP⁺ myotubes) where both Creand Lox-YFP myonuclei co-existed (FIG. 1B). No mononucleated cellsexpressing YFP were observed, thus confirming the validity of thislineage marking strategy. Adenoviral infection control was alsoperformed by co-culturing Ad-RFP infected MB with Lox-YFP MB indifferentiation medium (DM) and this did not yield YFP⁺ myotubes (FIG.1B). The YFP⁺ myotubes accounted for around 70% of total myotubes formedwithin 96 hours. Of these, around 60% of YFP⁺ myotubes, had 2-4myonuclei, while 30% had 5-7 myonuclei with an average number of 4.5myonuclei per YFP⁺ myotube. Non-YFP myotubes that arose from syngeneicfusion events of Cre-MB or Lox-YFP MB were also detected. This labelingstrategy was captured by time lapse microscopy encompassing total numberof 4 days from the co-culture of Cre and Lox YFP myoblasts to theirfusion into multinucleated myotubes Representative images of time lapsemicroscopy are shown in FIG. 10. To further rule out any possibility ofYFP expression without physiological myoblast fusion specific controlexperiments were conducted. The Cre-MB were co-cultured with Lox-YFP MBin mitogenic growth medium (GM; Ham's F10, 20% BGS, 9 ng/ml bFGF-2)where cells remained mononucleated and did not fuse into myotubes (FIG.1B). After 96 hours of co-culture in GM, cells were processed forwestern blotting (FIG. 1C), qRT-PCR analysis (FIG. 1D) andimmunostaining (FIG. 10) for YFP expression. No YFP expression wasobserved by any of these techniques. A more stringent control experimentwas performed to check the possibility of horizontal transfer of Crerecombinase, without complete fusion process. For this, Cre myoblastswere cultured in differentiation medium to form myotubes. Later, Lox YFPMB (2×10⁵ cells) were added to 96 hour old Cre expressing myotubes andcultures switched to mitogenic growth medium for 72-96 hours. As seen inFIG. 10E, both epifluorescent imaging and anti-YFP staining confirmedthe absence of YFP expressing mononucleated cells without physiologicalfusion of Cre-MB and Lox-YFP MB into myotubes. These results clearlyshow that indeed YFP⁺ myotubes arose only from the fusion of Cre andLox-YFP MB in DM. The results obtained and quantified with YFPlive-direct fluorescence were completely consistent with the dataproduced using anti-YFP immuno-fluorescence and both assays wereroutinely employed throughout these studies. The YFP⁺ myotubes werepositive for muscle differentiation markers myogenin and eMyHC, and forCDK inhibitor, p21 (which indicates the post-mitotic state) (FIG. 2A-C).Furthermore, these YFP⁺ myotubes did not incorporate BrdU, confirmingthat all YFP⁺ marked cells produced by fusion of Cre and Lox-YFP MB haveexited cell cycle and are in post-mitotic arrest by 96 hours in DM (FIG.2D). These findings establish an unambiguous lineage marking ofterminally differentiated myotubes that is dependent on physiologicalmyoblast fusion.

Example 2 Inhibition of Tyrosine Phosphatase and Apoptosis ReprogramsMyotubes into Proliferating Mononucleated Cells

Myotube formation involves many events such as changes in cytoskeletalassembly, sequential expression of differentiation specific genes,modulation of signaling pathways and up-regulation of tyrosinephosphatases (Bennett and Tonks, Science 278, 1288-1291, 1997; Delgadoet al., Genomics 82, 109-121, 2003; Lassar et al., Curr Opin Cell Biol6, 788-794, 1994; Weintraub, Cell 75, 1241-1244, 1993). Hence wereasoned that global transient inactivation of tyrosine phosphataseswould reset signaling in myotubes, making them receptive to mitogenspresent in growth medium conditions and propelling them into cell cycleas well as towards less differentiated state. Earlier BpV (a tyrosinephosphatase inhibitor) was reported to delay differentiation of dividingC2C12 into myotubes (Castaldi et al., FASEB J 21, 3573-3583, 2007) andother studies also indicated that a small percentage of myotubes thatenter S-phase upon over-expression of genes fail to proliferate andsuccumb to apoptosis (Endo and Nadal-Ginard, J Cell Sci 111 (Pt 8),1081-1093, 1998; Latella et al., Cell Death Differ 7, 145-154, 2000).Therefore, we reasoned that if BpV was able to trigger the process ofmyotube de-differentiation and that the addition of an apoptosisinhibitor (Q-VD) in our studies may help in survival of those myotubesthat might undergo massive restructuring of cell cytoskeletonsimultaneously with the breakage of post-mitotic arrest. Using ourlineage marking technique for myotubes, we then explored whetherBpV+Q-VD (henceforth inhibitor mix) was capable to reprogram alreadydifferentiated primary myotubes to their muscle progenitor fate. Forde-differentiation assays, inhibitor mix (10 μM each) was added to YFP⁺myotube cultures daily for two days after which cultures were switchedto GM (FIG. 3A). Remarkably, in the presence of inhibitor mix,considerable number of YFP⁺ myotubes showed altered morphology andcleaved into small cells which were followed by the appearance of YFP⁺mononucleated progeny of the de-differentiated myotubes (FIG. 3B). Whenmyotubes produced by the fusion of primary myoblasts were treated withBpV alone, cell death occurred as described in previous studies (Rumoraet al., 2003). Interestingly, in the presence of BpV alone, myotubes didshow apoptosis and few of them gave rise to YFP⁺ mononucleated cellsalbeit at very low frequency (˜1.18%) in comparison to inhibitor mixtreatment which augmented the de-differentiation frequency to around˜12%-13% (FIGS. 11A, 11C and 11D). These results demonstrate that theinhibition of apoptosis is a factor for the de-differentiation ofmultinucleated primary myotubes. In control experiments, no YFP⁺mononucleated cells were observed in untreated YFP⁺ myotubes that wereswitched to GM for 72 hours (FIG. 3C), or in the presence of Q-VD alone(FIG. 11B). Furthermore, the YFP⁺ cells derived from the post-mitoticmultinucleated myotubes engaged in proliferation, where ˜12%-13% oftotal lineage marked population was found to incorporate BrdU during a24 hour BrdU labeling interval (FIGS. 3D and 3E). The former myotubeidentity of these YFP⁺ cells clearly discriminated them from thereactivation of reserve YFP⁻ myoblasts, which also proliferated andexpanded when myotube cultures were switched to the highly mitogenic GM(FIG. 3D). To rule out any spurious YFP expression in the absence of Creexpressing cells and in the presence of inhibitor mix, 4 day old Lox YFPmyotube cultures were treated with the inhibitor mix and then switchedto growth medium. No YFP expression was ever observed in these cultures.This shows that Lox YFP cells do not spontaneously express YFP uponaddition of inhibitor mix in the absence of Cre recombinase (FIG. 13A).To capture reprogramming of lineage marked myotubes, we also performedtime lapse microscopy where Ad-Cre-Lox YFP myotubes were labeled for 96hours followed by the addition of inhibitor mix for another 48 hours andthe cultures were then switched to GM. Live cell imaging was performedfor the total period of 4 days where inhibitor mix treated YFP⁺multinucleated myotubes gave rise to YFP⁺ mononucleated cells.Representative images of the time lapse imaging can be seen in FIG. 12.A combination of a small molecule tyrosine phosphatase inhibitor, BpVand an apoptosis inhibitor, Q-VD was sufficient for such reprogrammingof terminally differentiated muscle cells. Since, inhibitor of tyrosinephosphatases induces apoptosis, the possibility that some aspect ofapoptosis may mediate the reprogramming of multinucleated myotubes toundergo de-differentiation was also examined. 0.2 uM doxorubicin, aclassic inducer of apoptosis which has been studied in muscle (Latellaet al., 2004) was added to Cre-Lox myotube cultures for 48 hours eitheralone or in combination with the apoptosis inhibitor. This was followedby removal of drugs and switching cultures to growth media conditionsfor 72-96 hours. We observed reduced apoptosis by doxorubicin in thepresence of apoptosis inhibitor but no YFP⁺ mononucleated cells wereobserved in these cultures in spite of altered morphology of myotubes(FIGS. 13B and 13C). These suggests that apoptosis does not mediate thede-differentiation of myotubes.

In parallel to these experiments, we also addressed a possible role ofOct4 in myotube reprogramming considering the pivotal reprogrammingactivity of Oct4 and the ability of this transcriptional factor alone toreprogram neural stem cells (Kim et al., Nature, 2009). We used ourpublished method (Conboy and Conboy, Methods Mol Biol 621, 149-163,2010) to activate endogenous muscle stem (satellite) cells by injuryinto hind limb muscle of Tet-Oct4 mice (Hochedlinger et al., Cell 121,465-477, 2005) and derived primary myoblasts which were kept in DM toform myotubes and then treated or untreated with doxycycline (dox) for24 and 48 hours, to induce Oct4 protein and mRNA expression (FIG. 14).These Tet-Oct4 myoblasts were infected with Ad-Cre and co-cultured withLox-YFP MB in DM for 96 hours, where myotubes were readily formed (FIG.14). No significant changes in morphology of myotubes were observed upto 48 hours of Oct4 induction followed by growth media incubation foradditional 4 days (FIG. 14D). In concert with earlier studies, where ithas been reported that induction of Oct4 in the differentiated cells ofthe intestine and hair follicle has no effect on their cellularphenotype (Hochedlinger et al., supra, 2005), our results showed thatOct4 induction up to 2 days was not sufficient to induce anymorphological changes in myotubes or to promote their de-differentiationin GM (FIG. 14D).

Example 3 Mononucleated Cells Generated from De-Differentiated MyotubesExhibit Hallmarks of Myogenic Progenitors

To further assess the properties of reprogrammed cells, mono-nucleatedYFP⁺ progeny of de-differentiated myotubes was FACS sorted and expandedin culture (FIGS. 15A and 15B). These YFP⁺ mononucleated cells wereimmunostained with antibody against Ki67 (proliferation marker) and BrdU(S phase marker) along with YFP. As shown in FIGS. 15C and 15D, theseFACS sorted expanded YFP⁺ cells positively immunostained for Ki67 andincorporated BrdU. Further, cell cycle analysis by propidium iodide DNAstaining confirmed that these cells can proliferate and exist indifferent phases of cell cycle (FIG. 15E). To confirm that the activelydividing reprogrammed YFP⁺ cells are indeed myogenic, they were analyzedfor the myogenic markers Pax7, MyoD1 and differentiation markersmyogenin, eMyHC and Cdk inhibitor, p21 (FIG. 4A). Based on thequantification of the immunofluorescence, around 70% of YFP⁺mononucleated cells expressed high levels of Pax7 and ≥90% expressedMyoD1 (FIG. 4B). The differentiation capability of the YFP⁺mononucleated cells was tested by switching the cultures to DM wherenormal primary myoblasts exit cell cycle and fuse into multinucleatedmyotubes. The YFP⁺ precursor cells were found to retain their myogenicpotential as they underwent rapid physiological fusion de-novo intomyotubes that expressed typical muscle differentiation markers, eMyHCand myogenin and p21 (FIG. 4C) Thus, the markers of terminaldifferentiation that were down-regulated upon myotube reprogramming withinhibitor mix treatment, were up-regulated again when YFP⁺ myogenicprogenitor cells differentiated into de-novo myotubes in the mitogen-lowdifferentiation medium (FIG. 4D). The changes in marker gene expressionwas also validated at transcriptional level by qRT-PCR which clearlyshowed the up-regulation of eMyHC, p21 and myogenin and down regulationof Pax7 and MyoD mRNA levels upon differentiation of YFP⁺ mono-nucleatedcells (FIG. 4D). Thus, based on the profile of myogenic markers and thefunctional properties, de-differentiated genetically labeled progeny ofprimary myotubes acquired the fate of muscle precursor cells ormyoblasts.

Example 4 Reprogrammed Progenitor Cells Contribute to In Vivo MuscleRegeneration

The ultimate test was to make sure that these reprogrammed cells couldcontribute to in vivo muscle regeneration under physiologicalconditions. The dividing de-differentiated YFP⁺ cells were expanded inGM for ˜1.5-2 weeks and injected into cardiotoxin injured TibialisAnterior (TA) of immuno-deficient NOD-SCID mice. Injections ofun-recombined Lox-YFP myoblasts and buffer medium served as negativecontrols. Two weeks of post injection, muscles were dissected out,sectioned and immunostained for YFP and laminin. YFP⁺ reprogrammed cellsreadily fused with regenerating myofibers and contributed to musclerepair in vivo (FIG. 5). These results establish that post-mitoticmyotubes can de-differentiate into functional, proliferating myogenicprecursor cells that regenerate muscle tissue after an injury. Culturedmuscle precursor cells that eagerly regenerate muscle in vivo wereproduced from terminally differentiated primary myotubes without anexogenous gene expression, making this method therapeutically feasible.

Example 5 Inhibitor Mix Treatment Modulates Gene Expression in Myotubes

To address the mechanism by which inhibitor mix facilitatedde-differentiation of YFP⁺ myotubes, we analyzed early changes inexpression of eMyHC (a terminal muscle differentiation marker), myogenin(a muscle marker expressed on onset of differentiation), p21, p15 andp16 (CDK inhibitors) in Ad-Cre-Lox YFP⁺ myotubes treated with inhibitormix for 48 hours (FIGS. 6A and 6B). By immunofluorescence, over 60% ofthe myonuclei in YFP⁺ myotubes down regulated myogenin as compared tountreated myotubes whereas the levels of p21, a negative regulator ofmitosis that plays an important role in cell-cycle arrest (Bunz et al.,Science 282, 1497-1501, 1998; Cayrol et al., Oncogene 16, 311-320, 1998)were found to be attenuated in approximately 25% of the myonucleipresent in YFP⁺ myotubes (FIGS. 6C and 6D). The down regulation ofeMyHC, p21, p15, p16 and myogenin in Ad-Cre-Lox-YFP⁺ myotubes treatedwith inhibitor mix was also confirmed by western blotting experimentsand qRT-PCR analysis (FIGS. 6E and 6F). These results demonstrate thatthe myogenic cell fate is reversed at the genetic level inmulti-nucleated myotubes, before they split into single dividing cells.We also observed that inhibitor treatment increased the expression ofYFP in Cre-Lox myotubes both at protein and RNA level. As shown earlierin FIG. 13A, Lox YFP myotubes do not express YFP spontaneously uponinhibitor mix treatment and in the absence of Cre recombinase. Howeverwhen YFP locus is recombined, the broad range phosphatase inhibitor mixwhich is known to modulate numerous signaling pathways by acting at thetranscriptional as well as post transcriptional levels might activateROSA locus at the transcriptional level thereby increasing YFPexpression.

Studies have also shown that upon myotube differentiation, widespreadchromatin remodeling occurs and genes necessary for differentiation areactivated while those for proliferation are repressed (Forcales andPuri, Semin Cell Dev Biol 16, 596-611, 2005; Guasconi and Puri, TrendsCell Biol 19, 286-294, 2009; McKinsey et al., Curr Opin Cell Biol 14,763-772, 2002; Palacios and Puri, J Cell Physiol 207, 1-11, 2006;Sartorelli and Caretti, Curr Opin Genet Dev 15, 528-535, 2005). Since asubset of labeled myotubes enter cell cycle and proliferate in GM, wereasoned that inhibitor mix have a global effect and perturbation ofsignaling pathways would in turn affect chromatin remodeling therebyfacilitating reprogramming of myotubes to their progenitor cells. Toanalyze these changes during inhibitor mix treatment, PCR arrays forchromatin enzymes and chromatin remodeling factors was performed onuntreated and inhibitor mix treated Cre-Lox YFP myotube cultures (FIG.7). As summarized in FIGS. 7C and 7D, Carm1, Suv39h1, SWI/SNF complexcomponents which have been earlier shown to promote myogenicdifferentiation (Ait-Si-Ali et al., EMBO J 23, 605-615, 2004; Chen S.L., et al., J Biol Chem 277, 4324-4333, 2002; de la Serna et al., NatGenet 27, 187-190, 2001) were down regulated upon inhibitor mixtreatment along with other histone methyltransferases. FIGS. 8 and 9show Supplementary Tables S1 and S2 that provide a complete list ofchromatin factor and enzyme genes modulated by inhibitor mix treatment.These findings suggest that inhibitor mix down-regulates the chromatinfactors and enzymes dedicated to the maintenance of differentiated statein primary myotubes, enabling them to respond to the growth factorspresent in serum and de-differentiate to YFP⁺ proliferating progenitorcells.

The studies presented herein explored the small molecule pharmacologicalapproach to de-differentiation and reprogramming that does not involveover-expression of exogenous genes, which is the currently searched formethod in the field of cell reprogramming. The use of a broadpharmacological inhibitor of tyrosine phosphatases simultaneously withthe inhibition of apoptosis was, in our hands, sufficient to induceactual reprogramming of terminally differentiated post-mitoticmultinucleated skeletal muscle cells into their progenitors. The use ofsmall molecule inhibitors for reprogramming studies has hightranslational significance. The apoptosis inhibitors used in our studiesis reversible treatment, has short half life and is not present in cellswhen expanded in vitro for transplantation experiments. Hence thereprogrammed myogenic progenitor cells transplanted with the aim toalleviate myopathic conditions will not be resistant to apoptosis andconsequentially will not pose risk of cancers. In this work theirreversible cell-fate lineage marking of myotubes was based on the factthat terminally differentiated skeletal muscle cells are normallyproduced by the fusion of myoblasts. Thus, Lox-YFP Rosa 26 nuclei andCre containing nuclei at some point coexisted in a multinucleated cellin order to produce YFP⁺ myotubes. The Cre-Lox myotubes labeling methodefficiently distinguishes reserve cells from multinucleated myotubes.Our data shows that 4 day old cultures of YFP⁺ primary myotubes expresstypical muscle differentiation markers such as myogenin, eMyHC, expresshigh levels of CDK inhibitor p21 and do not incorporate BrdU whichstrongly suggests that these YFP marked cells are indeed terminallydifferentiated (FIG. 2A-D). The observation that dividing YFP⁺mononucleated myogenic progeny were obtained from YFP⁺ myotubesunambiguously establishes the reprogramming step toward a lessdifferentiated precursor cell. Importantly, such genetic labeling forthe first time demonstrates de-differentiation of mature 4-day oldmultinucleated primary myotubes into proliferating fusion-competentmyoblasts that expand in vitro and repair muscle in vivo.

The calculation of reprogramming efficiency from terminallydifferentiated myotubes to the muscle progenitor cells is complicated bythe fact that heterogenous Cre and Lox YFP myoblasts form YFP⁺ myotubeswhere varying number of both myonuclei co-exist in the samemultinucleated myotube. Further, only Lox YFP myonuclei and not Ad-CreMB nuclei co-existing in YFP⁺ myotubes will give rise to YFP⁺mono-nucleated cells when labeled myotube de-differentiates. Moreover,the de-differentiation of myotubes that are produced by syngeneic fusionevents was not accounted for in our YFP labeling method. Hence, weestimated efficiency by two different methods. By method 1 total numberof YFP⁺ mononucleated cells was divided by the total number of YFP⁺myotubes before inhibitor treatment. Based on this method, efficiencywas estimated ˜12-13% in the presence of inhibitor mix as compared toBpV alone which was around ˜1.18% (FIG. 11D). By method 2, the totalnumber of YFP⁺ mononucleated cells was divided by an estimated number ofLox YFP myonuclei present in all labeled YFP⁺ myotubes before inhibitortreatment. This method gave an estimate of ˜5% in presence of inhibitormix and ˜0.4% in presence of BpV alone (FIG. 11D). For the abovereasons, we feel these calculations give very conservative estimates ofde-differentiation. No matter, the method of quantification, BpV alonegave poor reprogramming efficiency and apoptotic inhibitor was needed toaugment the de-differentiation likely by facilitating the survival ofthose myotubes which undergo reprogramming in the presence ofphosphatase inhibitor.

Recent reports have shown that cells expressing higher levels of antiproliferative genes and those involved in senescence are indeeddifficult to reprogram (Li et al., Nature 460, 1136-1139, 2009; Utikalet al., Nature 460, 1145-1148, 2009). Since myotubes arepost-mitotically arrested cells which express high levels of CDKinhibitors, these may have low reprogramming efficiency. Studies haveindicated that experimental down regulation of CDK inhibitors inpost-mitotic myotubes results in accumulation of DNA damage, hinderscell cycle reentry and cause DNA fragmentation and apoptosis (Pajalungaet al., PLoS ONE 5, e11559, 2010). It has been shown that dividing cellsrobustly repair DNA damage (Nouspikel and Hanawalt, DNA Repair (Amst) 1,59-75, 2002) and since our de-differentiated cells are cultured inmitogen-high growth medium (following the BpV and apoptosis inhibitortreatments) and are actively dividing, any DNA damage accumulated inmyotubes is likely to be repaired during the process of DNA replication.Notably, our results definitively demonstrate that reprogrammed myotubesgive rise to functional muscle progenitor cells which form new muscle invitro and in vivo, hence no irreversible DNA damage or mutationsoccurred to compromise the myogenic properties of the de-differentiatedcells. Recent studies has also shown that inhibition of Rb and p16/p19can induce cell cycle entry in post mitotic myocytes (Pajcini et al.,Cell Stem Cell 7, 198-213, 2010). Our work conducted on 4 day old maturemyotubes not only down regulated CDK inhibitors and muscledifferentiation markers but also decreased gene expression of chromatinremodelers that maintain differentiated state. The role of chromatinorganization in establishment and maintenance of cell fates has beenwell defined. It has also been shown to play a role in commitment ofmyoblast to terminally differentiated myotubes as different signalingpathways have been shown to modulate chromatin signaling in muscleprogenitor cells upon differentiation (Caretti et al., Genes Dev 18,2627-2638, 2004; de la Serna et al., Nat Genet 27, 187-190, 2001;McKinsey et al., 2002; Palacios and Puri, J Cell Physiol 207, 1-11,2006). Recent work has also demonstrated the stage specific role of Ezh2in muscle regeneration where Ezh2 occupies the Pax7 regulatory sequencesin differentiated state (Palacios et al., Cell Stem Cell 7, 455-469,2010). This suggests that in spite of down regulation of Ezh2 upondifferentiation as reported earlier (Caretti et al., Genes Dev 18,2627-2638, 2004), a certain level of Ezh2 along with other polycombmembers would be required to maintain muscle progenitor genes inrepressed state. Our PCR arrays on Cre-Lox myotubes after inhibitor mixtreatment demonstrate significant down regulation of Ezh2 and otherpolycomb members as compared to untreated myotubes suggesting thecreation of sensitized background in myotubes that may force them tore-express muscle progenitor genes and enter cell cycle.

Since a generic tyrosine phosphatase and apoptosis inhibitor were usedwhich is expected to modulate signaling in many biochemical pathways,the down-regulation of specific chromatin remodeling factors predisposedprimary myotubes to respond to the mitogens of GM thereby changing theircell fate to that of proliferating myogenic precursor cells.Interestingly, while upon addition of inhibitor mix there were profoundchanges in the morphology of many myotubes, only a subset of thesemyotubes de-differentiated into proliferating mono-nucleated precursorcells. Since there is temporal progression towards the degree ofterminal differentiation in myotubes there might be a specific timewindow when myotubes would be more responsive to the treatment andcapable of undergoing cell fate reversal. Future work can determine theexact role of chromatin remodeling factors in acquiring muscleprogenitor cell fate from multinucleated post-mitotic differentiatedstate. As there is a temporal progression of gene expression uponmyotube differentiation, it would be interesting to study in the futurewhether the inhibitor mix is sufficient to induce de-differentiation inmature myofibers formed in vivo or indeed other factors are required toyield regenerative cells.

Use of pharmacological inhibitors to modulate different signalingpathways without gene over expression is therapeutically relevant incoaxing differentiated cells to yield regenerative cells. Our datahighlights the combinatorial use of tyrosine phosphatase and apoptosisinhibitors for primary myotube de-differentiation to yield myogenicproliferating cells which aided in muscle regeneration both in vitro andin vivo in SCID mice. The novel myotube labeling technique developed forthis study served as a powerful tool to clearly show the origin ofreprogrammed cells and to sort them away from reactivated reservemyoblasts. Importantly, small molecule inhibitors induced changes inmyotubes at epigenetic level and facilitated them to enter proliferativestate in mitogen rich medium. All together, the novel labeling techniqueemployed along with the use of small molecule inhibitors advance theongoing research in regenerative medicine and would enable uniqueclinical strategies for enhancing tissue regeneration.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

What is claimed is:
 1. A method of screening for an agent that inhibitstyrosine phosphatases and an agent that inhibits apoptosis and mediatesgeneration of lineage-restricted progenitor cells from differentiatedcells, the method comprising: lineage marking differentiated cells in acell population by labeling the differentiated cells such thatlineage-restricted progenitor cells generated from the differentiatedcells are labeled and can be distinguished from other cells in the cellpopulation; contacting the cell population comprising the labeleddifferentiated cells with an effective amount of the candidate agentthat inhibits tyrosine phosphatases and an effective amount of thecandidate agent that inhibits apoptosis of the differentiated cell;isolating the labeled lineage-restricted progenitor cells that aregenerated from the labeled differentiated cells, if present, from theother cells in the cell population; and determining whether the labeledlineage-restricted progenitor cells are generated from the labeleddifferentiated cells, wherein the differentiated cells and thelineage-restricted progenitor cells have the same lineage, wherein thepresence of the labeled lineage-restricted progenitor cells indicatesthat the candidate agent that inhibits tyrosine phosphatases and thecandidate agent that inhibits apoptosis mediate generation oflineage-restricted progenitor cells from the differentiated cells. 2.The method of claim 1, wherein the differentiated cells are myocytes andthe lineage-restricted progenitor cells are myogenic progenitor cells.3. The method of claim 2, wherein the myocytes are myocytes selectedfrom the group consisting of cardiomyoctytes, smooth muscle myocytes,and skeletal myocytes.
 4. The method of claim 1, wherein thedifferentiated cells are from a subject with a disease.
 5. The method ofclaim 4, wherein the subject is in need of tissue regeneration.
 6. Themethod of claim 5, wherein the subject is suffering from loss of musclefunction and/or loss of muscle mass.
 7. The method of claim 1, whereinthe candidate agent that inhibits tyrosine phosphatase is potassiumbisperoxo(1,10-phenanthroline)oxovanadate (bpV(phen)).
 8. The method ofclaim 1, wherein the candidate agent that inhibits apoptosis isN-(2-Quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methyl ketone(Q-VD-oPh).
 9. The method of claim 1, wherein the labeled differentiatedcells are genetically labelled.
 10. The method of claim 1, wherein thelabeled differentiated cells are labeled with a Cre-Lox method.
 11. Themethod of claim 1, wherein the contacting is carried out ex vivo. 12.The method of claim 1, wherein the isolating comprises flow cytometry.13. The method of claim 12, wherein the flow cytometry comprisesfluorescence-activated cell sorting (FACS).
 14. The method of claim 1,wherein the determining comprises measuring gene expression.
 15. Themethod of claim 1, wherein the determining comprises quantitativereverse transcription polymerase chain reaction (qRT-PCR).
 16. Themethod of claim 1, wherein the determining comprises western blotting.17. The method of claim 1, wherein the method further comprisestransferring the lineage-restricted progenitor cells to conditions thatpromote the differentiation of the lineage-restricted progenitor cellsto differentiated cells of the same lineage as that of the labeleddifferentiated cells contacted in the contacting step.
 18. The method ofclaim 17, wherein the transferring comprises transferring thelineage-restricted progenitor cells into a subject in need of tissueregeneration.
 19. The method of claim 18, wherein the subject issuffering from loss of muscle function and/or loss of muscle mass. 20.The method of claim 1, wherein the candidate agent that inhibitstyrosine phosphatases is a vanadium compound, and the candidate agentthat inhibits apoptosis is a caspase inhibitor.